System and method to simulate hemodynamics

ABSTRACT

A system for hemodynamic simulation comprises a vessel having properties of a blood vessel, a reservoir containing a quantity of fluid, tubing connecting the vessel and reservoir, and at least one pump for circulating the fluid within the system. Fluid can be tissue culture medium or blood analog fluid, and the vessel may include mammalian cells attached to its inside. A drive system, comprising two reciprocating drive shafts that are coupled by a cam, enables the uncoupling of pulsatile flow and pulsatile pressure to provide independent control over wall shear stress and circumferential strain. The shaft drives two pumps that are 180 degrees out-of-phase and are connected upstream and downstream of the vessel, and effect this uncoupling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. §119(e), of U.S.Provisional Application No. 60/239,015, filed Oct. 6, 2000 by theapplicant, and which is herein incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work described in this application was supported by funding from theNational Institutes of Health under Grant No. HL-35549. The UnitedStates Government may have certain rights to the invention.

FIELD OF THE INVENTION

The present invention is a system and method for simulating thehemodynamic patterns of physiologic blood flow. In particular, thepresent invention can simultaneously generate wall shear stress andcircumferential strain patterns relevant to cardiovascular function anddisease.

BACKGROUND OF THE INVENTION

Cardiovascular disease is the leading cause of death in the UnitedStates, and costs millions of dollars per year. Atherosclerosis is theleading cause of death in the developed world and nearly the leadingcause in the developing world. Atherosclerosis is a disorder in whichthe coronary arteries become clogged by the build up of plaque along theinterior walls of the arteries, leading to decreased blood flow whichcan in turn cause hypertension, ischemias, strokes and, potentially,death.

Atherosclerosis has been shown to occur in sites of complex hemodynamicbehavior. Surgical intervention is often employed to treat it, and mayinclude insertion of a balloon catheter to clean out the plaque, andinsertion of a stent within the vessel to enable it to remain open, ormay include multiple bypasses of the clogged vessels. Bypass surgeryinvolves the removal of a section of vein from the patient's lower leg,and its transplant into the appropriate cardiac blood vessels so thatblood flows through the transplanted vein and thus bypasses the cloggedvessels.

A major problem associated with bypass surgery is the patency of thevessels to be used in the bypass. The bypass vessels are prone tofailure, which may occur within a short period of time after bypasssurgery, or after a period of several years. Hemodynamic forces havebeen implicated as a major factor contributing to the failure of thebypass vessels.

Hemodynamic forces (i.e., forces due to blood flow) are known toinfluence blood vessel structure and pathology. The vascular cellslining all blood vessels are endothelial cells, which are importantsensors and transducers of the two major hemodynamic forces to whichthey are exposed: wall shear stress (“WSS”), which is the fluidfrictional force per unit of surface area, and hoop stress, which isdriven by the circumferential strain (“CS”) of pressure changes. Wallshear stress acts along the blood vessel's longitudinal axis.Circumferential strain is associated with the deformation of the elasticartery wall (i.e., changes in the diameter of the vessel) in response tothe pulse of vascular pressure. Wave reflections in the circulation andthe inertial effects of blood flow cause a phase difference, the stressphase angle (“SPA”), between CS and WSS. The SPA varies significantlythroughout the circulation, and is most negative in disease pronelocations, such as the outer walls of a blood vessel bifurcation.Hemodynamic forces have been shown to dramatically alter endothelialcell function and phenotype (i.e., high shear stress [low SPA] isassociated with an atheroprotective gene expression profile, and a lowshear stress [large SPA] is associated with an atherogenic geneexpression profile). There is thus a great need to study vascularbiology in a complete, integrative, and controlled hemodynamicenvironment.

Despite the significance of hemodynamic WSS and CS acting on the vesselwall, especially at regions of the circulation with a high risk oflocalization of cardiovascular diseases, detailed knowledge of thecombined influence of the time varying patterns of WSS and CS onendothelial cell biological response has remained technologicallyunfeasible.

Laboratory studies of vascular fluid mechanics have demonstrated thatwall shear stress (WSS) and circumferential strain (CS) are out of phasetemporally, and that there is a systematic variation of the stress phaseangle (SPA) throughout the circulation. This variation is highlyout-of-phase in the large arteries, where arterial disease generallyoccurs, while in the smaller vessels and veins where disease is rare,this variation is generally in phase.

Where an artery bifurcates, Spa varies with the local spatial positionwithin that bifurcation, the more out-of-phase environment beinglocalized on the outer wall of the bifurcation where atherosclerosisoccurs. SPA was found to be more out-of-phase in the coronary arteriesthan at any other location in the circulation.

Prior technology has focused on the individual effects of WSS or CS,individually, on endothelial cells. Berthiaume and Frangos described adevice that simulates wall shear stress using a rod and plate systemthat is similar to the cone and plate system used in viscometers. Changdescribed a parallel flow chamber used to simulate steady flow. Carosiet al and Sumpio et al. describe devices to simulate cyclic strain thatconsists of a flexible membrane that is stretched by a motor or a vacuumsuction system.

Qiu and Tarbell described a device to simulate pressure and flow intubes, but the device did not permit using a wide range of phase angles(SPAs), and was technically difficult to use. Limitations, however, ofthe Qiu and Tarbell system included having the maximum attainable phaseangle being 100 degrees, the amplitude and phase of the flow andpressure are coupled, and the system utilized large quantities of fluid.The present invention, by its selection of tubing and vessel diameters,in contrast, employs approximately one fifth the volume of fluid as thatsystem. Seliktar et al. in an in vitro study, verified that simulationof the hemodynamic environment is critical to vessel patency andfunction.

The patent literature described several systems for examining theeffects of strain, or the effects of shear, individually, on cells orblood vessels.

Seliktar et al. (U.S. Pat. No. 5,928,945) describes a bioreactor forproducing cartilage in vitro, comprising a growth chamber, a substrateon which chondrocyte cells or chondrocyte stem cells are attached, andmeans for applying relative movement between a liquid culture medium andthe substrate to provide a shear flow stress to the cells attached tothe substrate.

In U.S. Pat. No. 5,899,937 Goldstein et al. describe a closed, sterilepulsatile loop for studying tissue valves. The system provides a tool toexamine heart valve leaflet fibroblast function and differentiation asthese are affected by mechanical loading, as well as an apparatus toprovide heart valves seeded with suitable cells. The sterile pulsatileflow system which exposes viable tissue valves to a dynamic flowenvironment imitating that of the aortic valve.

Wolf et al. (U.S. Pat. No. 5,271,898) discloses an apparatus for testingblood/biomaterials/device interactions and characteristics, comprising astepper-motor driven circular disc upon which a test vehicle is mounted.The test vehicle comprises a circular, closed loop of polymer tubingcontaining a check valve, and contains either the test materials,coating, or device. The apparatus generates pulsatile movement of thetest vehicle. Oscillation of the test vehicle results in the pulsatilemovement of fluid over its surface.

In U.S. Pat. No. 6,205,871 B1 Saloner et al disclose a panel ofanatomically accurate vascular phantoms comprising a range of stenoticconditions varying from normal to critically stenosed (0% area reductionto greater than 99% reduction), and which phantoms are subjected topulsatile flow of a blood mimic fluid.

Vilendrer (U.S. Pat. No. 5,670,708) discloses a device for measuringcompliance conditions of a prosthesis under simulated physiologicloading conditions. The prosthesis includes stents, grafts andstent-grafts, which is positioned within a fluid conduit of theapparatus, wherein the fluid conduit is filled with a saline solution orother fluid approximating the physiological condition to be tested. Thefluids are forced through the fluid conduit from both ends of theconduit in a pulsating fashion at a high frequency simulating systolicand diastolic pressures.

In U.S. Pat. No. 4,839,280 Banes describes an apparatus for applyingstress to cell cultures, comprising at least one cell culture platehaving one or more wells thereon, with each of the wells having asubstantially planar base formed at least partially of an elastomericmembrane made of biocompatible polyorganosiloxane composition, with theelastomeric membrane having an upper surface treated to permit cellgrowth and attachment thereto by means of the incorporation at the uppersurface of a substance selected from the group consisting of an amine, acarboxylic acid, or elemental carbon, and vacuum means for controllingthe elastomeric membrane to the pulling force of a vacuum. Banes (U.S.Pat. No. 6,218,178 B1) discloses an improvement, in the form of aloading station assembly for allowing stretching of a flexible cellculture membrane, the assembly comprising a planar member and a postextending from a surface of the planar member, an upper surface of thepost being configured to support a flexible cell culture membrane, theplanar member defining a passageway configured to allow fluid to flowthrough from one side of the planar member to an opposite side of theplanar member, and wherein the flexible cell culture member isstretchable at a periphery of the upper surface towards the planarmember.

In U.S. Pat. Nos. 4,940,853 and 5,153,136 Vanderburgh describes a methodand apparatus for growing tissue culture specimens in vitro,respectively. The apparatus comprises an expandable membrane forreceiving a tissue specimen thereon, a mechanism for expanding themembrane and the tissue specimen, and a controller for controlling theexpanding mechanism. The controller is operative for applying anactivity pattern to the membrane and a tissue specimen thereon whichincludes simultaneous continuous stretch activity and repetitive stretchand release activity. The continuous stretch and release activitysimulate the types of activity to which cells are exposed in vivo due togrowth and movement, respectively, and they cause the cells of tissuespecimens grown in the apparatus to develop as three-dimensionalstructures similar to those grown in vivo.

In U.S. Pat. Nos. 5,217,899 and 5,348,879 Shapiro et al. describe anapparatus and method for stretching cells in vitro, respectively. Theinventions impart to a living culture of cells biaxial mechanical forceswhich approximate the mechanical forces to which cells are subjected invivo. The apparatus includes a displacement applicator which may beactuated to contact and stretch a membrane having a living cell culturemounted thereon. Stretching of the membrane imparts biaxial mechanicalforces to the cells. These forces may be uniformly applied to the cells,or they may be selectively non-uniformly applied.

Lee et al. (U.S. Pat. No. 6,057,150) discloses a biaxial strain systemfor cultured cells that includes a support with an opening over which anelastic membrane is secured, a moveable cylinder coaxial with theopening and fitting closely but movably within the opening, and anactuating member that stabilizes and controls the position of thecylinder relative to the opening. The actuating member is coupled to thesupport by a threaded connection while engaging the movable cylinder.The degree of membrane stretch is accurately controlled by the rotationof the actuating member.

In U.S. Pat. No. 4,851,354 Winston et al. disclose an apparatus formechanically stimulating cells, comprising an airtight well having anoptically transparent compliant base of a biologically compatiblematerial on which the cells may be grown and an optically transparent,removable cap, coupled with a ported, airtight reservoir which reservoirhas an optically transparent base and which reservoir can be filled withpressuring media to create cyclic variations in hydrostatic pressurebeneath the complaint base, causing the compliant base to deform andthereby exert a substantially uniform biaxial force on the cellsattached thereto.

Lintilhac et al. (U.S. Pat. No. 5,406,853) disclose an instrument forthe application of controlled mechanical loads to tissues in sterileculture. A slider which contacts the test subject is in forcetransmitting relation to a forcing frame. Tension, compressive andbending forces can be applied to the test subject, and force applied tothe test subject is measured and controlled. A dimensionalcharacteristic of the test subject, such as growth, is measured by alinear variable differential transformer. The growth measurement datacan be used to control the force applied. Substantially biaxialstretching is achieved by placing the test subject on an elasticmembrane stretched by an arrangement of members securing the elasticmember to the forcing frame.

In U.S. Pat. No. 6,107,081 Feeback et al. disclose a unidirectional cellstretching device capable of mimicking linear tissue loading profiles,comprising a tissue culture vessel, an actuator assembly having arelatively fixed structure and an axially transformable ram within thevessel, at least one elastic strip which is coated with an extracellularmatrix, and a driving means for axially translating the ram relative tothe relatively fixed structure, and for axially translating the endportion of the elastic strap affixed to the ram relative to another,opposite end portion, for longitudinally stretching the elastic strap.

Nguyen et al. (U.S. Pat. No. 5,272,909) disclose a method and device fortesting venous valves in vitro. The device comprises (a) a fixture formounting a sample valve on a liquid flow path. (b) a muscle pumpcomponent and/or (c) respiratory pump component and/or (d) capacitancereservoir component and/or (e) vertical hydrostatic column component,all of the components being fluidly connected to the flow path to mimicthe muscle pump, respiratory pump, capacitance and hydrostatic impedanceeffects of actual in situ venous circulation in the mammalian body. Themuscle pump is designed to mimic effects caused by movement of thevisceral organs and somatic muscles on a vein, while the respiratorypump is designed mimic the effects of normal cyclic variations in theintra-thoracic pressure due to the movement of the thoracic muscles anddiaphragm. The combination of pumps of the present invention provides ameans to examine the effects of pulsatile pressure, wall shear stress,and circumferential strain, separately or in combination, on bloodvessels or mammalian cells in vitro.

In U.S. Pat. No. 5,537,335 Antaki et al. disclose a fluid deliveryapparatus in which a predetermined pressure waveform is introduced intoa conduit, such as a human saphenous vein. By such exposure, the veincan be “arterialized”, meaning that it can be conditioned in preparationfor its use in bypass surgery, an excised vein according to theinventors. The combination of pumps and the manner of controlling thedegree of their being in phase or out-of-phase with each other providesa means to examine not only the effects of a blood pressure waveform,but also the effects of pulsatile pressure, wall shear stress, andcircumferential strain, separately or in combination, on blood vesselsor mammalian cells in vitro.

The most common WSS simulating systems utilize a 2-dimensional stiffsurface, such as a glass slide, for the endothelial cell culture formingthe wall of a parallel plate flow chamber. The WSS in these devices isusually steady because of difficulties in simulating pulsatile flow.Cyclic straining devices provide only strain, by stretching cells on acompliant membrane without flow. Both types of systems are thus limitedby their design. However, no studies have been performed studying bothparameters (WSS and CS) using cells grown on a single type of supportsurface because such a system, until now, has remained technologicallyunfeasible. The present invention addresses and solves this long-feltneed by providing a system in which endothelial cells can be grown on asingle support surface, and subjected to studies in which both wallshear stress and circumferential strain can be examined independently ofeach other.

The use of a silicone tube coated with endothelial cells was recentlyintroduced, and provided the potential for simultaneous coupledpulsatile strain and shear stress. However, these tubes were used inflow simulators coupling pressure and flow that could only achieve phaseangles (SPAs) of about 90–100 degrees; such a phase angle was inadequatefor simulating coronary arteries, the most disease prone vessels in thecirculation, because coronary arteries are characterized by a high SPA,on the order of approximately 250 degrees. These flow simulators weredifficult to use and to produce replicable reliable results. The presentinvention overcomes this problem, by providing time-varying uniformcyclic pressure (and consequently CS) and pulsatile flow (andconsequently WSS) in a 3-dimensional configuration over a complete rangeof SPAs, as a most complete physiologic environment.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a system to simulatephysiological hemodynamics.

Another object of the present invention to provide a system to simulatebiomechanical stimuli due to fluid flow, pressure and pressuredifferentials (transmural pressure).

Another object of the present invention is to provide a system in whichthe effects of wall shear stress (“WSS”) and circumferential strain(“CS”) can be studied independently of each other.

Another object of the present invention is to provide a system in whichthe effects of wall shear stress (“WSS”) and circumferential strain(“CS”) can be studied simultaneously.

Another object of the present invention is to provide a system in whichthe effects of wall shear stress (“WSS”) and circumferential strain(“CS”) can be studied independently of each other over a wide range ofstress phase angles (“SPA”).

Another object of the present invention is to provide a system in whichthe effects of vasoactive compounds can be studied.

Another object of the present invention is to provide a system in whicheffects of vasoactive compounds can be studied on the genes thatregulate their production.

It is an object of the present invention to provide a system to simulatephysiological hemodynamics of a plurality of blood vessels.

It is an object of the present invention to provide a system to simulatephysiological hemodynamics of a plurality of mammalian blood vessels.

It is an object of the present invention to provide a system to simulatephysiological hemodynamics of a plurality of human blood vessels.

It is an object of the present invention to provide a method for tosimulating physiological hemodynamics.

Another object of the present invention to provide a method ofsimulating biomechanical stimuli due to fluid flow, pressure andpressure differentials (transmural pressure).

Another object of the present invention is to provide a method forstudying effects of wall shear stress (“WSS”) and circumferential strain(“CS”) independently of each other.

Another object of the present invention is to provide a method for thesimultaneous study of the effects of wall shear stress (“WSS”) andcircumferential strain (“CS”) on vessels.

Another object of the present invention is to provide a method for theindependent study of the effects of wall shear stress (“WSS”) andcircumferential strain (“CS”) over a wide range of stress phase angles(“SPA”).

Another object of the present invention is to provide a method forstudying the effects of vasoactive compounds.

Another object of the present invention is to provide a method forstudying the effects of vasoactive compounds on the genes that regulatetheir production.

It is an object of the present invention to provide a method forsimulating physiological hemodynamics of a plurality of blood vessels.

It is an object of the present invention to provide a method forsimulating physiological hemodynamics of a plurality of mammalian bloodvessels.

It is an object of the present invention to provide a method forsimulating physiological hemodynamics of a plurality of human bloodvessels.

The present invention achieves the uncoupling of pulsatile flow andpulsatile pressure to provide independent control over WSS and CS. Thesystem at first seems paradoxical since it is classically well knownthat pressure and flow are coupled. However, in a dynamic sinusoidalenvironment, such as that of the present invention, flow and pressurecan be independently modulated and therefore, appear to be uncoupled.The drive system, comprising two reciprocating drive shafts that arecoupled via a circular cam effects this uncoupling. The flow shaftdrives pumps, that are at opposite ends, that are 180 degreesout-of-phase and are connected to the recirculating flow loop upstreamand downstream of the test section (compliant vessel). The flow shaftallows independent control of pulsatile flow with no pulsatilecircumferential strain. The second (pressure) shaft also drives twopiston pumps that are 180 degrees out-of-phase; however, one pistondrives the internal pressure upstream to the test section and the otherpiston drives the external chamber pressure. The pressure shaft allowsfor independent control of the pulsatile pressure. The attachment pointsof the circular cam that couples the two drive shafts can be adjusted toprovide the phase (between 0 and 360 degrees) between the motions of thetwo shafts. This phase difference provides simulation of a wide range ofSPAs, including the disease prone coronary arteries (approximately 250degrees). Since the flow is related to wall shear stress (WSS) and thepressure is related to the circumferential strain (CS), the pulsatileWSS and pulsatile CS are independent and uncoupled.

The present invention is a system for hemodynamic simulation comprisinga vessel having properties of a blood vessel, a reservoir containing aquantity of fluid, tubing connecting the vessel and reservoir, and atleast one pump for circulating the fluid within the system. Fluid can betissue culture medium or blood analog fluid, and the vessel may includemammalian cells attached to its inside. A drive system, comprising tworeciprocating drive shafts that are coupled by a cam, enables theuncoupling of pulsatile flow and pulsatile pressure to provideindependent control over wall shear stress and circumferential strain.The shaft drives two pumps that are 180 degrees out-of-phase and areconnected upstream and downstream of the vessel, and effect thisuncoupling.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1A is a top plan schematic view of the hemodynamics simulator ofthe present invention.

FIG. 1B is a side view illustrating the 4-bar linkage of the presentinvention.

FIG. 1C is a more detailed schematic diagram of the embodiment of FIG.1A.

FIG. 1D is a schematic diagram of an embodiment which includes a bypassof the compliant vessel.

FIG. 2 is a plot of the diameter (circles) and pressure (triangles)waveforms as a function of time with a zero degree stress phase angle(SPA) difference.

FIG. 3 is a plot of the diameter (triangles), pressure (crosses) andflow (squares) waveforms as a function of time with a sixty degreestress phase angle (SPA) difference.

FIG. 4 is a plot of the diameter (squares), pressure (triangles) andflow (diamonds) waveforms as a function of time with a ninety degreestress phase angle (SPA) difference.

FIG. 5 is a plot of the diameter (squares), pressure (triangles) andflow (diamonds) waveforms as a function of time with a one hundredeighty degree stress phase angle (SPA) difference.

FIG. 6 illustrates the structure of the support and support mount.

FIG. 7 illustrates the shape of the support rod.

FIG. 8 a and b illustrates fluid flow through the support rod and vesselusing different shaped support rods. The arrow in Panels A and Brepresents the direction of fluid flow:

-   -   Panel A: using a linear shaped support rod;    -   Panel B: using a tapered support rod.

FIG. 9 a and b illustrates another embodiment of the noise filter(vibration damper). Panels A and B represent two differentconfigurations.

FIG. 10 is a schematic diagram of a second embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION.

The present invention is a hemodynamic simulator 10, shown schematicallyin FIG. 1A, and in greater detail in FIG. 1B. The hemodynamic simulator10 comprises a sample chamber 12 (which will also be referred to hereinas “compliant vessel”) which may comprise either a non-rigid tube thatcontains mammalian cells, a blood vessel excised from a mammal, or otherbiocompatible substrate containing cells or onto which cells can begrown or attached thereto. Sample chamber 12 is connected to a reservoir14 containing an appropriate fluid 16, which may comprise a tissueculture medium, blood or a blood analog fluid, physiological salinesolution (generally a solution of 0.9% sodium chloride ((“NaCl”)), asknown to those skilled in the art), or other buffered solution.

Reservoir 14 generally is a sterilizable container comprising aplurality of fittings 20 which function to provide, for example only andnot intended as any limitation except as described in the claims,temperature probe insertion; pH probe insertion; inflow and outflow ofculture medium 16; inflow and outflow of one or more gases, such as, butnot limited to, CO₂, nitrogen, oxygen, air or other gas or gaseousmixture, such as 5% CO₂ in air; as may be required; media sampling port;addition of acid, base or other buffering agent for the adjustment orother control of medium pH. Reservoir 14 is generally made of a standardlaboratory grade glass, but, as known to those skilled in the art, mayalso comprise any type of sterilizable plastic vessel which can meet thesystem's requirements.

The system 10 includes a first pump 22, which is generally used toprovide a steady flow of fluid 16 through the system, such that fluid 16flows between reservoir 14 and compliant vessel 12 through tubing 24. Inone embodiment of the present invention, the flow rate is maintained asa steady rate, controlled by first pump 22. In this embodiment, firstpump 22 is a centrifugal pump, such as one the Biomedicus 520d(manufactured by Biomedicus Corp., Minneapolis, Minn.). In anotherembodiment of the present invention, first pump 22 is a peristalticpump, such as that sold by MasterFlex Corp., New Brunswick Scientific(New Brunswick, N.J.) or other commercial laboratory supplymanufacturers. Other types of pumps can also be employed as first pump22, such as a DISC-FLO® pump, a gear pump, or other pumps which mustprovide a constant volumetric flow.

In the embodiment wherein the first pump 22 is a peristaltic pump, anoise filter 26 is required, in order to dampen the noise (highfrequency vibrations) created by the movements of the peristaltic pump(FIG. 1B). The noise filter may also be referred to herein as a pulsedamper, and is commercially available from laboratory supply houses,such as the PULSE DAMPENER® (Cole-Parmer Corp., Vernon Hills, Ill.). Thenoise filter 26 also serves as a bubble trap, preventing the passage ofbubbles that may be generated by the pump. As will be described infurther detail below, the system may also include a bypass to preventbubbles from entering the compliant vessel (see FIG. 1C).

An alternate embodiment of the noise filter 26 is illustrated in FIG. 9,the differences between the noise filter in FIGS. 9A and 9B being theconfiguration of the container 72. Container 72 comprises a inlet 74 andoutlet 76 ports for the inflow and outflow of fluid 16 from the system,respectively. Air inlet 78 and outlet 80 ports are also fitted into thecontainer. In addition, a pressure relief valve (not shown) can befitted into container 72. The alternate embodiments of the noise filterreduce the amount of fluid required by the system, compared to theamount of fluid used when the commercial noise filter is employed.

Generally, it is preferred to utilize a minimal amount of fluid 16 inorder to reduce the costs of media utilization, drug treatment, and cellby-product (such as, but not limited to, proteins, metabolites and like)detection and the like. In the embodiment shown in FIGS. 1A–1C,approximately 100 ml of fluid are employed. The length of the tubingfrom the vibration damper 26 to the upstream connector also providesadditional high frequency steady flow pump induced vibration damping.

Tubing 24 generally comprises any suitable type of laboratory tubingwhich is capable of being sterilized. Such tubing includes that soldunder the trademark of Tygon® (Norton Co., Worcester, Mass.); PharMed®tubing (Trademark of PharMed Group Corporation, Miami, Fla.), siliconetubing, or other comparable laboratory or medical-surgical tubing fromother manufacturers.

The length of the upstream tubing is chosen so as to minimize the totalvolume of fluid used in the system. Its length is calculated to providea maximum flow rate, and to avoid turbulence in the system, based uponboundary layer theory, as known to those skilled in the art, anddescribed further below.

The compliant vessel 12 is supported proximate its ends 28, 30 by a pairof supports 32 which are held in place by a pair of rigid mounts 34,respectively. The mounts 34 and supports 32 preferably are as shown inFIGS. 6-8, each mount including an opening 62 therethrough, toaccommodate a support 32 therein. To facilitate the alignment of thecompliant vessel 12 within the support system, a support rod 64 isinserted into aperture 66 located on each support mount 62. A set screw68 may be used to retain the support rod 64 in position. The supportmount 34 preferably is made from a non-corrosive, durable material, andcapable of withstanding autoclaving; stainless steel is one suchmaterial. Each support 32 comprises a tube having ends 70 shaped to fitthe ends 28, 30 of compliant vessel 12 (FIGS. 8A and 8B). As shown inFIG. 8B, the tapered end 70 of support 32 provides a fit at the ends ofcompliant vessel 12 such that there is a negligible disturbance of fluidflow, in contrast to the disturbance that would occur if the end ofsupport was linear (FIG. 8A). The ends of the compliant vessel 12 areattached to each support using clamps, suturing, or other methods knownto those skilled in the art. In one embodiment of the present invention,the supports 32 are manufactured from TEFLON® (polytetrafluorethylene,DuPont Co., Wilmington, Del.) or stainless steel, but other suitable,biocompatible materials can be substituted.

Depending upon the which properties (WSS, CS, pressure) are to bestudied, the compliant vessel 12 may be surrounded by an externalchamber 36, but external chamber is not required under allcircumstances. In such instances, the external chamber is opened to theatmosphere. External chamber 36 is a sealed chamber that has a port withwhich the chamber can be filled with a fluid such as water or otherfluid, and a second port through which contents of the chamber 36 can bepressurized by connection to one of the pumps 42. External chamber 36may also be a jacketed chamber, enabling a cooled or heated fluid tocirculate around the compliant vessel 12 in order to maintain thetemperature required by the contents of the compliant vessel 12, and thechamber connected to a circulating bath, such as those manufactured bythe Neslab Corporation.

Although not essential to the operation of the hemodynamic simulator 10of the present invention, an additional length of tubing 24 can be addedto function as a compliant vessel bypass 38 (FIG. 1C). The bypass tubing38 is connected both upstream and downstream of the compliant vessel 12,so that if problems occur when the system is started from a zero flowrate and pressure to the desired flow and pressure, such as bubbleformation, the bypass can be used until proper conditions are achieved,at which point the bypass 38 is closed off or removed, and flow isresumed through the compliant vessel 12.

The support 32 is made from tubing having an inner diameter (I.D.) thatmatches the I.D. of both the compliant vessel 12 and the upstreamtubing. By having the I.D. of the support matching the I.D. of thevessel and tubing, this prevents flow separation and an underdevelopedflow regime from occurring. The wall of the support 32 should taper tothe outside such that the compliant vessel 12's I.D. does not bendabruptly as it is placed over the support. This provides a flush I.D.surface between the support 32 and the compliant vessel 12 and greatlyminimizes flow separation. One possible configuration is to have theupstream tubing, the support 32 and the compliant vessel 12 to be madeof one piece with a rigid structure around the upstream end and support.

Drive System

The system further comprises a plurality of pumps 40 and 42, furtherdesignated as second pumps 40 (also referred to herein as P1 and P2),and third pumps 42 (also referred to herein as P3 and P4), respectively(FIGS. 1A and 1B). As shown in FIG. 1A, pumps P1 and P3 are connected tothe “upstream” flow of the hemodynamic system 10 of the presentinvention, pump P2 is connected to the “downstream” flow, and pump P4 isconnected to the external chamber 36, providing external pressure on thecompliant vessel 12 contained therein. Fluid 16 or the like flowsdownstream back into reservoir 14, in a closed flow system; the culturefluid is recycled to conserve culture fluid, but if the culture fluidbecomes unsuitable for growth, such as caused by acid build-up therein,reservoir 14 can be replaced with one containing a fresh quantity offluid 16, as appropriate. The various components of the presentinvention are connected by sterile fittings, and components can bechanged, aseptically, as experimental or other conditions so require.

Each of pumps 40 and 42 is under the control of a drive system unit 44,which comprises a plurality of independent linear actuators 46. Theseactuators 46 can be individual, stand alone units, for may be controlledby one or more computer systems 48. In the embodiment in FIG. 1A, thesecond pumps 40 are connected by a shaft 50, and the third pumps 42 areconnected by a second shaft 52. In one embodiment of the presentinvention, in which a 4-bar linkage mechanism is the drive system, a cam54 affects the control of the various second pumps 40 and third pumps42. In one embodiment of the present invention (FIG. 1B) the drivesystem unit 44 comprises six computer-controlled linear actuators, whilein another embodiment (FIG. 1A) the drive system unit 44 comprises fourindependent computer-controlled linear actuators.

The hemodynamic simulator 10 includes a plurality of sensors 18 formeasuring hemodynamic parameters. These sensors 18 include a flowsensor, which may be placed either upstream and/or downstream of thecompliant vessel 12. Such a flow sensor can be an ultrasound Dopplerprobe, as known to those skilled in the art. The Doppler probe,depending upon its position in the system, can either be a sterileprobe, and/or a probe that may or may not be fluid-contacting. Anelectromagnetic probe may also be used as a flow sensor. In oneembodiment of the present invention, the flow sensor is an ultrasonicflow meter (Transonics Systems, Inc.) which is positioned in-line andjust upstream of the compliant vessel. Flow rate variation over thelength of the compliant vessel has been negligible.

A pressure sensor 18 is used for monitoring the internal systempressure, and positioned either upstream and/or downstream of thecompliant vessel 12. A pressure sensor can also be placed in theexternal chamber 36 to monitor external chamber pressure. Pressuresensor 18 can also be a blood pressure catheter (such as, for example,and not intended as a limitation, a MILLAR catheter (MPC-500 withpressure meter TCB500; Registered Trademark of Millar Instruments Corp.,Houston Tex.), in either a fluid contacting or non-contacting version.Pressure sensor 18 may also be a pressure probe, such as those known tothose skilled in the art. In one embodiment of the present invention,the pressure sensor is a catheter tip transducer (Millar) which isinserted upstream into the lumen of the compliant vessel. Where cellsare being used in the compliant vessel 12, the pressure sensor 18 iskept upstream to avoid damaging the cells. Pressure drop across thecompliant vessel has been shown to be negligible.

The linear actuators 46 may be selected from among those that comprise acam mechanism; a multi-bar linkage mechanism, such as an actuatorcomprising a four-bar mechanism; a solenoid; a stepper motor; anelectric motor, whether operated by alternating current (“AC”) or directcurrent (“DC”); a linear ball actuator; a belt driven actuator; a chaindriven actuator; or any other drive unit which is capable of producing avariable cyclic motion, or any combination of the above actuators, suchas, for example only, and not intended to be a limitation, thecombination of a cam mechanism and a 4-bar linkage mechanism and a DCmotor. The cyclic motion generated by the drive system unit can resemblethat of a blood pressure waveform in its magnitude, frequency and otherproperties, as known to those skilled in the art. By adjustment of thedrive system components, as known to those skilled in the art, theextent of the phase differences among the second pumps 38 (P1–P4) can beadjusted, from anywhere between 0 degrees and 360 degrees.

It has been classically known to those skilled in the art that pressureand flow are coupled, and could not be uncoupled. Using the dynamicsinusoidal environment created by the hemodynamics simulator 10 of thepresent invention, flow and pressure can be uncoupled.

This uncoupling is achieved using the drive system 44 of the presentinvention, comprising two reciprocating drive shafts 50 and 52 that arecoupled via a circular cam 54 (FIG. 1A). Each flow shaft 50 or 52 drivestwo piston pumps P1 and P2, or P3 and P4, respectively (at oppositeends) that are 180 degrees out-of-phase and are connected to therecirculating flow loop upstream and downstream of the compliant vessel12 (test section). The flow shaft allows independent control ofpulsatile flow with no pulsatile circumferential strain. The second(pressure) shaft 52 also drives two piston pumps that are 180 degreesout-of-phase; however, one piston drives the internal pressure upstreamto the compliant vessel 12 (test section) and the other piston drivesthe external chamber pressure. The pressure shaft allows for independentcontrol of the pulsatile pressure. The attachment points of the circularcam 54 that couples the two drive shafts can be adjusted to provide thephase (between 0 and 360 degrees) between the motions of the two shafts.This phase difference provides simulation of a wide range of SPAs,including the disease prone coronary arteries (approximately 250degrees). Since the flow is related to wall shear stress (WSS) and thepressure is related to the circumferential strain (CS), the pulsatileWSS and pulsatile CS are independent and uncoupled. In this process,changes in the upstream pressure may have an effect on the downstreampressure, such that if the stroke of the upstream pumped is changed, thestroke of the downstream pump does require compensation.

Prior to setting up the hemodynamic simulator 10 of the presentinvention, system components are sterilized. Sterilization can beeffected, depending upon the components of the system, by methods suchas autoclaving, ethylene oxide (EtO) treatment, ultraviolet lightirradiation, gamma irradiation, and other methods known to those skilledin the art.

The hemodynamic simulator 10 is generally run at a temperature ofapproximately 37 degrees Centigrade, but it can be operated attemperatures ranging from approximately 20 degrees Centrigrade toapproximately 50 degrees Centigrade. As shown in FIG. 1B, the “testsection”, representing the compliant vessel 12, and support means 32 and34 can be immersed in a water bath 56 of the appropriate temperature.The hemodynamic simulator 10 can be operated for a duration ranging fromas short as a few minutes, for example, 5–10 minutes, to more extendedlengths of time, such as, between approximately 72 hours to 168 hours.In a preferred situation, the hemodynamic simulator is operated over aperiod of between approximately 5 hours and approximately 72 hours. Alimiting factor in the duration of the hemodynamic simulator 10'soperation is maintenance of sterility of the system.

It is to be understood that factors such as the geometry of the vessel,the diameter of the vessel, the viscosity of the medium used, thepressure, and the flow rate of the medium through the vessel, are amongthe factors that determine the wall shear stress (WSS), and that whenreference is made to WSS, these factors are taken into consideration.

By insertion of the compliant vessel 12 within the external chamber 36,the effects of diameter variation, caused by circumferential strain andwall shear stress, can be studied, in the absence of pulsatile pressure(condition 2).

The diameter variation of the compliant vessel is measured using adiameter sensor. The diameter sensor can be a non-contacting ultrasoundtransducer 82 (such as a single element transducer V312 101.25 andpulser-receiver unit 5072, both from Panametrics Co., Waltham, Mass.,not shown). The ultrasound probe position must be perpendicular to andaligned with the center of the diameter of the test specimen in order tosense the diameter. One beam passes through the specimen (a pulse),differences in material densities results in peaks and beam profilealterations that are detected with the receiver, and are subsequentlyacquired and processed using a computer which includes an oscilloscopewith peak detection software and appropriate analytical software. Alinear cross-sectional profile of the specimen is then detected,providing the dimensions of the outer and inner walls, and consequently,wall thickness. The probe can be positioned anywhere in the test sectionto provide dimensions. Absolute and relative dimensions can be obtained,for example, relative dimensions are sufficient for monitoring diametervariations. The dimensions are monitored and acquired, via the computer,in real-time along with pressure, flow and other measurements. Amulti-array ultrasound probe can also be used to monitor diametervariation. The diameter sensor can also utilize lasers, video imaging,magnetic resonance imaging, other imaging modalities, or can be acontacting probe, such as known to those skilled in the art.

All data signals are acquired by the computer system, which is not shownin the drawings. The ultrasound diameter monitoring requires a peakdetection algorithm. Phase angle is determined using Fast FourierTransforms (“FFT”). Some signals are used for monitoring, and feedbackcontrol such as mean pressure, is monitored and adjusted via a motorcontrolled downstream reactor.

The wall shear stress waveform is determined based on the measured flowwaveform and the mean diameter according to Womersley (1955, andincorporated hereinby reference).

Initially, the flow is run at a low flow rate, and then the flow isadjusted to a high flow rate. The resistor 58 is adjusted to provide amean pressure, and the oscillatory drive system unit 44 is engaged tooscillate the ends of the sample, depending upon the experimentalconditions under investigation, by varying the movement of second pumps40, (P1 and P2) and third pumps 42 (P3 and P4). The resistor 58 is adevice that controls the degree of occlusion of the downstream flow toachieve a desired mean pressure. Examples of resistors suitable for usein the present invention include a gear motor controlled clamp devicethat controls occlusion of the downstream tubing; valves, pinch clampsor other types of laboratory clamps.

The hemodynamic simulator 10 of the present invention can simulate theimportant features of the mammalian hemodynamic environment,

The first hemodynamic conditions to be discussed are the fluid flow,pressure, and diameter variation (circumferential strain). The fluidflow and pressure (and consequently diameter variation) can bemanipulated to allow for precise control of the cyclic pulsatile fluidflow and pressure magnitude and phase. The fluid flow and pressure, andconsequently, the diameter variation in the case of tubular geometry,can be manipulated to allow for precise control of the cyclic pulsatilefluid flow and pressure magnitude and phase. A “tubular geometry case”,as used herein, is intended to refer to the use of curved vessels (forexample, half a toroid), bifurcated vessels (including variation such asbranched, Y-shaped, T-shaped, and the like). In other instances, thevessels employed are linear and non-branched.

There are several possible system configurations available, dependingupon the simulation conditions.

Complete control of the fluid flow and pressure relations attainableare:

-   -   Condition 1-fluid flow and pressure magnitude and phase (0–180        degrees) [i.e., wall shear stress 10 dynes per square centimeter        +/−10 dynes per square centimeter and 8% diameter variation with        their phase variation (angle) at 180 degrees for a compliant        vessel 12 made of silicone;    -   Condition 2-pulsatile flow and no pulsatile pressure (diameter        variation), magnitude and phase;    -   Condition 3-pulsatile pressure (diameter variation) and no        pulsatile flow magnitude and phase; and    -   Condition 4-pulsatile flow and pulsatile pressure (no diameter        variation) magnitude and phase.

In a compliant vessel where the transmural flux (hydraulic conductivityand/or permeability) can be monitored, conditions 1 and 2 require nochange or considerations. Condition 3 requires consideration of thepotential transmural reflux due to active transmural pressuremodulation. Condition 4 requires consideration of potential externalpressure augmentation due to increased hydraulic conductivity and/orpermeability that can be compensated for via an external pressurefeedback control mechanism.

Under Condition 1, the following combinations of second pumps 40 (P1 andP2), and third pumps 42 (P3 and P4) can be utilized: a) all four pumps,P1, P2, P3 and P4; b) P1, P2 and P4; or c) P1 and P3; or d) P2 and P4.

Under Condition 2, second control pumps 40, P1 and P2 are utilized.

Under Condition 3, third pumps 42, P3 and P4 are utilized.

Under Condition 4, second pumps 40 (P1 and P2) and third pumps 42 (P3and P4) are utilized.

The conditions are chosen according to the desired hemodynamicenvironment under simulation. Condition 1 is the most physiologicallyprevalent condition. The upstream, downstream, and external pressuresare modulated, primarily, with respect to amplitude, phase, andfrequency to achieve the desired hemodynamic environment. Theseparameters are effected using the controls of the drive system unit, alaboratory computer system 48.

The system thus operates with one of the second pumps 40 (in thisinstance, pump P1) affecting the upstream portion of the compliantvessel 12, and exerting its actions in a “pushing” manner along thecompliant vessel 12. A similar action is obtained with the third pump 42(pump P3) acting on the upstream end of compliant vessel. In contrast,the other of the second pumps 40 (in this instance, pump P3) affects thedownstream portion of the compliant vessel 12. Third pump P4 exerts anexternal pressure on the compliant vessel 12. The different actions ofthe pumps affect the movement/pulsation of the compliant vessel 12.

The effects of wall shear stress (WSS) are studied when the upstreamsecond pump P1 and the downstream third pump P3 are engaged. In thissituation, these pumps are working against each other by being 180degrees out of phase, and the upstream pump P1 causes an increase in theflow rate, while the downstream pump P3 causes a decrease in flow rate,resulting in no external pressure, and a combination of shear stress andpulsatile fluid flow through the compliant vessel 12.

When the hemodynamic simulator 10 of the present invention is used forstudying the effects of circumferential strain (CS) on the compliantvessel 12, one second pump, P1 and third pump P4, are used. In thissituation, the first pump 22 (the steady flow pump) can be shut off, andsecond pump P1 provides the upstream pressure, while third pump P4provides the external pressure on the compliant vessel 12.

The novel part of the apparatus is the drive system which induces thesinusoidal flow component and the diameter variation. In one embodimentof the present invention, the drive system 44 is a 4-bar linkagemechanism, shown schematically (FIG. 1). The second pumps 40 (P1 and P2)are connected by a first linkage 102. Third pumps 42 (P3 and P4) areconnected by a second linkage 104. Each linkage connects to piston 106of each pump. The linkages are connected to cams 54 by shafts 50 and 52,and each cam 54 is connected at 108 to a DC motor 110. Each drive shaft52, 54, is connected by an adjustable pivot 112, which adjusts thelength of the stroke of each pumps' piston 106. The drive systemcomprises two reciprocating drive shafts which are coupled through acircular cam. The phase between the motion of the two shafts can bevaried by adjusting the angle between the attachment points of the twoshafts on the common cam 54 (for example, zero degrees for in-phase, 180degrees for out-of-phase). One of the shafts 50 drives two piston pumpswhich are 180 degrees out-of-phase and are connected to therecirculating flow loop upstream and downstream of the compliant vessel12. The second shaft 52 drives two piston pumps which are also 180degrees out-of-phase; one pump feeds the flow loop upstream of thecompliant vessel, the second pump drives the external chamber. The twoout-of-phase piston pumps driving the internal flow loop act in apush-pull fashion. When the external chamber 36 is open to theatmosphere (when the second drive shaft 52 is disconnected) and thestroke volumes of the push-pull pumps on the first drive shaft areequal, a sinusoidal flow is generated, but with negligible pressurevariation because of the push-pull action. When the system is run inthis fashion (second shaft disconnected) it is possible to avesinusoidal flow (superimposed on the steady flow) with negligiblepressure or diameter variation. To induce diameter variation, the secondshaft is connected at any desired phase relative to the first shaft byadjustment of the cam 54. When both piston pumps on this shaft areinterfaced to this system, it is possible to adjust their stroke volumesso that the pressure in the external chamber and in the elasticcompliant vessel are nearly constant (as a result of the push-pullaction), and there is diameter variation driven by the volume changebetween the elastic compliant vessel and the external chamber (one fillswhile the other empties). When the system is run in this fashion, thereis sinusoidal flow with defined diameter variation and phase anglerelative to flow, but there is negligible pressure variation. Thisenables the present invention to uncouple pressure and stretch.

To introduce pressure variation in phase with diameter variation, whichis considered to be the most physiological condition, the drive line tothe external chamber is disconnected, and the chamber is left open tothe atmosphere. In this mode of operation, both pressure and diametervariation are driven by the upstream piston pump P3 on the second shaft50. Some interaction occurs between the pumps driven on the two shafts,but the volume flows driven by the second shaft 50 (controlling diametervariation) are very small compared to those driven by the first shaft 52(which controls flow), and they can be adjusted nearly independently.

The present invention was designed to overcome the current technologicallimitations in vascular research by physically simulating the normal anddiseased physiologic states. The present invention achieves a preciseand complete physiologic environment by uncoupling the major hemodynamicforces, WSS and CS, thereby permitting independent control over themagnitude and phase of the pulsatile WSS and CS to achieve a wide rangeof SPA. The present invention experimentally simulates real hemodynamicpatterns, both simple and complex patterns, while maintaining sterilityof the system, and employing a minimal volume of media demanded by celland tissue culture systems.

The advantage of cell and tissue culture systems is that the tools ofcell and molecular biology are easily employed. This integrativeapproach to the design of the present invention resulted in a systemthat is quick and easy to assemble and disassemble while maintaining thecell culture integrity that is important for biological assays. The testchamber of the present invention facilitates the insertion and removalof the test specimens. The test specimens are generally endothelial cellcoated silicone elastic tubes which are placed in the hemodynamicsimulator of the present invention, and yield biological resultsrelevant to the normal and diseased cardiovascular system.

Those skilled in the art have classically considered it well known thatpressure and flow are coupled. However in the dynamic sinusoidalenvironment, established by the invention, flow and pressure can beuncoupled, thereby providing independent control over WSS and CS.

The present invention not only provides a means for studyinghemodynamics in normal and diseased states, but it also can be used intissue engineering, to test or train the function of bypass vesselsprior to their use in coronary bypass surgery, or to investigatecryopreserved vessels for research or medical use. Current coronarybypass surgery most often utilizes vessels from the hemodynamicallyunstrenuous saphenous vein (in the lower leg) as the bypass vessel. Thepresent invention can be used to train the vessel to the strenuoushemodynamic environment of the coronary arteries. As can be seen fromthe foregoing, these applications are ultimately related to thetreatment of cardiovascular disease.

The present invention may also be useful for analysis of bone mechanics,and effects of flow and related parameters on the development ofosteocytes, chondrocytes and the like. Shear stress is known to increasethe production of types II and I collagen, and other extracellularproducts, thus potentiating the fact that further mechanical stimuli,such as strain and shear stress, would further improve production ofextracellular products. Stem cells can be stimulated to differentiate bymechanical stimuli, such as shear stress, strain, or solute transportsystems. Other applications include, but are not intended to be limitedto, effects on cell and tissue culture, tissue engineering, effects incomplex artery geometries, effects on cardiac valves and their in vitroevaluation, evaluation and standardization of imagery diagnostic methodsusing vascular phantoms, effects of pharmacological agents on cells andtissues, materials testing in standard environments and in microgravityenvironments, and on cells co-cultured in a mixed bioreactor.

EXAMPLE 1 Preparation of Silicone Tubing for Attachment and Growth ofEndothelial Cells

In this example, the vessel chosen for growth of endothelial cells is asilicone tubing, sold by Dow-Corning, Midland, Mich. under the brandname of SYLGARD 184 elastomer, or Silastic (MDX4-4210), Medical Gradetubing, and used to prepare elastic artery models. These models wereprepared using the method described by Lee and Tarbell (1997, and herebyincorporated by reference), and included the preparation of models ofhuman linear and bifurcating arteries.

For the preparation of linear elastic vessels, a pair of symmetric,half-cylindrical grooved molds made of a plastic, such as PLEXIGLASS,are machined to have a diameter that matches the inner diameter of theelastic model described above. In one preferred embodiment, the linearelastic vessels have a length of approximately 29 centimeters and aninner diameter of approximately 0.79 centimeters, in another embodimentof the present invention, vessels having a length of approximately 15 cmare employed. A solid wax, cylindrical core is prepared by distributingmelted wax (CARBOWAX®, Union Carbide Co.) into the mold, and placing themold inside another cylindrical mold of the same plastic; in thepreferred embodiment, this second mold has a diameter of approximately0.95 centimeters, so as to produce an annular layer having a diameter ofapproximately 0.080 centimeters. A solution of SYLGARD 184® and a curingagent, prepared in accordance to methods known to those skilled in theart, is poured into this part of the mold, vacuum deaerated by methodsknown to those skilled in the art, and then cured. After curing, theelastic vessel is removed from the mold.

The elastic vessels are treated to promote cell attachment before beinginoculated with cells. Briefly, the vessels are hydrophyllized in a 70%sulfuric acid solution, boiled in distilled water and then sterilized byautoclaving. The vessels are then coated with a layer of fibronectin (30micrograms/ml in Modified Eagle's Medium ((“MEM”)), a tissue culturemedium known to those skilled in the art, fibronectin is obtained fromcommercial sources).

While vessels having inner diameters ranging from between 1–10 mm can beused, vessels having an inner diameter of approximately 8 mm (0.79) cmhas been shown to be an optimal inner diameter, and allow for the use ofmultiple tubes in the present invention while keeping the overall sizeof the present invention, and the consumption of cell culture media andother expendibles, within a range that is manipulable by laboratorypersonnel. In the system shown in FIGS. 1A–1C, approximately 100 ml offluid are employed. Each end of the vessel is inserted into position inthe present invention as has been previously described, using thesupports 32 and mounts 34. Where necessary, sterile tubing connectorsare also employed to enable tubing and other components to be connectedinto the system under aseptic conditions.

EXAMPLE 2 Tissue Culture Conditions

Endothelial cells (“ECs”) were obtained either from bovine aortas(“BAECs”), or from human umbilical veins (“HUVECs”), and cultured bygrowth as primary cultures, using procedures described in Sill et al.(1995). the contents of which is hereby incorporated by reference.

The BAECs were the cells most commonly used with the present invention.An inoculum of between 60,000–80,000 cells per square centimeter is usedtwice, once to enable the cells to adhere to the surface of the vesselfor a 45 minute time period, and a second time after rotating theposition of the vessel 180 degrees to enable the vessel's other side tobecome coated. The cells are grown in a monolayer until confluency isachieved, in a 37 degree centrigrade tissue culture incubator in anatmosphere of 5% CO₂ in air. The preferred growth medium 16 isDulbecco's Modified Eagle's Medium (“DMEM”, obtained commercially fromSigma Chemical Corp., St. Louis, Mo.), containing 10% Fetal Bovine Serum(“FBS”, obtained commercially), 1% L-glutamine and 1% antibiotics(penicillin-streptomycin solution. For experiments, the medium comprisedDMEM without FBS, and 1% bovine serum albumen (“BSA”) and 1% antibiotics(penicillin-streptomycin solution; BSA and the antibiotics arecommercially available from Sigma Chemical Corp.). MEM (also obtainedfrom Sigma) may be employed, depending upon the type of cells beingutilized. Generally, the pH of the culture fluid is maintained atapproximately pH 7.2, +/−0.05, but a pH in the range betweenapproximately 7.0 to approximately 7.5 is acceptable.

Requirements of the fluid 16 include having a viscosity that can beelevated to achieve conditions of physiologic stress at modest flowrates. Dextran is used within the fluid while the present invention usesvessels of approximately 0.79 cm diameter; in instances employingvessels of smaller diameter, addition of dextran is not necessary. Thefluid should be free of Phenol Red and serum so as not to interfere withmeasurements of other cellular products, such as prostacycline or nitricoxide. Serum and other substances can be added to the media if thesesubstances are under study, or if the serum or substance is required bythe cell line.

In addition to the use of tissue culture media, other physiologicalfluids, such as blood from a mammal such as sheep, cow, pig, rabbit, orhuman cord blood or human blood, can be utilized. Artificial or analogblood fluids can also be used. Among the blood analog fluids known tothose skilled in the art is an admixture of glycerol in water, andadjusted to have a viscosity comparable to blood.

EXAMPLE 3 Effect of Different Stress Phase Angles: Zero Degree SPA

FIG. 2 is a plot of the diameter (circles) and pressure (triangles)waveforms as a function of time with a zero degree stress phase angle(SPA) difference.

Changes in the diameter of the compliant vessel 12 can be measured byone of several methods known to those skilled in the art. These includethe use of such non-contacting methods as ultrasound or laser light, orthe use of an elastic strain gauge, which is in physical contact withthe specimen (the compliant vessel). In the present invention, thepreferred method of monitoring the changes in compliant vessel diameteris with an ultrasound transducer (Panametrics Co., not shown) which ismounted through the exterior chamber wall and which is focused on thecompliant vessel.

The computer controlled drive unit 44 is capable of generating differentwaveforms, which can range from a sine wave, as employed in this and thesubsequent examples (FIGS.2–6), or which can be a blood pressurewaveform, such as a known waveform taken from a reference text, ordetermined experimentally on a human. For convenience in establishingthe parameters of the present invention, sine waves were chosen. Theflow waveform represents the rate of flow of the culture medium 16 orother fluid through the system as a function of time. The flow rates, inmilliliters per minute, have been normalized so as to fit on a scaleranging from plus 1 to minus 1. Similarly, data representing thepressure on the compliant vessel 12, expressed in mm of mercury, and thedegree of distortion of the diameter of the compliant vessel (diameterwaveform) have also been so normalized.

The rate of wall shear in the compliant vessel was measured using aphotochromic method of flow visualization for use in elastic tubes.Using a focused laser beam having a specific wavelength, the laser beampasses through the vessel, containing a photo-sensitive dye of acorresponding wavelength, and causes the dye to change color andgenerate a dye line within the fluid flow. Using a video camera torecord the displacement of the dye line caused by the pulsating laserbeam, the near wall velocity profile form which the wall shear rate canbe determined from the slope at the wall, using methods described inRhee and Tarbell (1994, and incorporated by reference herein). In thisexample, the preferred laser is a nitrogen laser with a wavelength inthe range of the ultraviolet (VSL337ND, from Laser Science Inc.).

A polyalkylene glycol ether, described in Weston et al. (1996, andincorporated by reference herein) would be usable because this agent hasthe Theological properties comparable to blood, and the photodynamicproperties that are compatible with the material from which thecompliant vessels were manufactured.

FIG. 2 illustrates that when there is no difference in the phase anglebetween the flow and the pressure, the pressure waveform and thediameter waveform are similar to each other.

EXAMPLE 4 Effect of Different Stress Phase Angles: Sixty Degree SPA

FIG. 3 is a plot of the diameter (triangles), pressure (crosses) andflow (squares) waveforms as a function of time with a sixty degreestress phase angle (SPA) difference.

When the phase angle between the flow and the pressure are sixty degreesout of phase, the pressure waveform and the diameter waveform remainsimilar to each other, while the flow waveform is shifted (FIG. 3).

EXAMPLE 5 Effect of Different Stress Phase Angles: Ninety Degree SPA

FIG. 4 is a plot of the diameter (squares), pressure (triangles) andflow (diamonds) waveforms as a function of time with a ninety degreestress phase angle (SPA) difference.

When the phase angle between the flow and the pressure are ninetydegrees out of phase, the pressure waveform and the diameter waveformremain similar to each other, while the flow waveform is shifted (FIG.4).

EXAMPLE 6 Effect of Different Stress Phase Angles: One Hundred EightyDegree SPA

FIG. 5 is a plot of the diameter (squares), pressure (triangles) andflow (diamonds) waveforms as a function of time with a one hundredeighty degree stress phase angle (SPA) difference.

When the phase angle between the flow and the pressure are one hundredeighty degrees out of phase, the pressure waveform and the diameterwaveform remain similar to each other, but the flow waveform is shiftedto an even greater extent compared to when they are either 60, or 90ninety degrees out of phase (compare FIG. 5 with FIGS. 2–4).

EXAMPLE 7 Compliant Vessels

Example 1 described the use of vessel models, modeled after thestructure and material properties of actual human aortic vessels. Inaddition to using models of vessels, other vessels can be used inconjunction with the present invention. These can be chosen from thegroup consisting of an artery, an artificial artery, a vein, humanumbilical tissue, or a nonrigid tube. The artery may comprise a bovineaorta, or a human coronary artery. The vein may comprise bovine veins,or human veins such as a human leg vein or a human umbilical vein.Bovine tissue can be obtained from commercial supply sources, such asVec Technologies, Ithaca N.Y. and human umbilical materials can beobtained a local hospital, or a commercial sources such as Clonetics,Vec Technologies, or other sources known to those skilled in the art. Inaddition to studying the effects of hemodynamic conditions onendothelial cells, other types of cells can also be used, includingsmooth muscle cells, cartilage cells, osteocytes, embryonic and adultstem cells, and the like.

The tubing employed as the vessel can have any geometry, ranging fromgeometries, such as, for example only and not intended as anylimitation, straight, curved, bifurcating, branched or the like. Thevessel may also be chosen from any chamber, whether having a parallelflow, a radial flow, etc. The vessel may also be made of any material,such as, but not limited to, materials such as silicone, collagen, anartery, a vein, glass, tissue culture grade plastics or the like; suchmaterials are considered to be biocompliant. The compliant vessel canthus have any combination of these properties.

EXAMPLE 8 An Embodiment for Studying Hemodynamics on Multiple Vessels

In this embodiment of the present invention (shown schematically in FIG.10, and in which like reference numerals refer to like elements), thehemodynamics simulator 200 can be used to study hemodynamic propertiesof a plurality of compliant vessels 12. This embodiment is similar tothat described in FIGS. 1A and 1B, but comprises a plurality ofcompliant vessels 12, a plurality of reservoirs 14, a first pump 22which has been adapted to pump fluid through a plurality of tubing 24,and a plurality of noise filters 26, as needed, as has been describedfor that embodiment (FIG. 1B). The compliant vessels 12 are enclosed ina plurality of external chambers 36. Under such conditions, compliantvessels 12 can be studied with and/or without an external chamber 34under otherwise comparable experimental conditions. The drive systemunit 44 is similar to that described previously (FIGS. 1A–1B). Althougha plurality of reservoirs 14 are illustrated in FIG. 10, a singlereservoir could be used to supply all of the compliant vessels 12, ormultiple reservoirs containing different types of culture media or otherbiological fluid 16, could be used, for examining the effects of eitherdifferent cell types under identical stress conditions, or the effectsof different fluids on a cell line, or other combinations desired to beexamined by one skilled in the art.

Therefore, although this invention has been described with a certaindegree of particularity, it is to be understood that the presentdisclosure has been made only by way of illustration and that numerouschanges in the details of construction and arrangement of parts may beresorted to without departing from the spirit and scope of theinvention.

REFERENCES

-   Berthiaume, F., Frangos, J. A. 1993. “Flow effects on endothelial    cell signal transduction, function and mediator release.”    Flow-dependent regulation of vascular function. Bevan et al., Oxford    Univ. Press, New York.-   Carosi, C. G., Eskin, S. G., and McIntire, L., 1992. Cyclic strain    effects on production of vasoactive materials in cultured    endothelial cells. J. Cellular Physiol. 151:29–36.-   Lee, C. S., and Tarbell, J. M. 1997. Wall shear rate distribution in    an abdominal aortic bifurcation model: Effects of vessel compliance    and phase angle between pressure and flow waveforms. J. Biomech.    Engr. 119:333–342.-   Rhee, K., and Tarbell, J. M. 1994. A study of the wall shear rate    distribution near the end-to-end anastomosis of a rigid graft and a    compliant artery. J. Biomechanics 27:329–338.-   Qiu, Y. C., and Tarbell, J. M. 2000. Interaction between wall shear    stress and circumferential strain affects endothelial cell    biochemical production. J. Vascular Res. 37:147–157.-   Seliktar, D., Nerem, R. M. et al. 2000. Dynamic mechanical    conditioning of collagen gel blood vessel constructs induces    remodeling in vitro. Ann. Biomedical Eng. 28:351–362.-   Sampio, B. E., and Widmann, M. D. 1990. Enhanced production of    endothelial-derived contracting factor by endothelial cells    subjected to pulsatile stretch. Surgery 108:277–282.-   Weston, M. W., Rhee, K., and Tarbell, J. M. 1996. Compliance and    diameter mismatch affect the wall shearrate distribution near an    end-to-end anastomosis. J. Biomechanics 29:187–198.-   Womersley J. R. 1955. Method for the calculation of velocity, rate    of flow and viscous drag in arteries when the pressure gradient is    known. J. Physiol. 127:553–563.

All patents and references cited herein are hereby incorporated byreference in their entirety.

1. A system for hemodynamic simulation, the system comprising: a fluid;a vessel through which the fluid may be urged; a chamber in which thevessel is received, the chamber including a means for controllingpressure; a reservoir for retaining the fluid; a plurality of pumps influid communication with the fluid, one of the pumps urging the fluidthrough the vessel; and a means for controlling the pumps, wherein themeans for controlling the pumps comprises a motor, a cam, and a meansfor linking the pumps, wherein the pumps are operatively connected tothe means for controlling the pumps.
 2. The system as described in claim1, wherein the means for linking the pumps is adjustable, and whereinthe pumps are out of phase with each other.
 3. The system as describedin claim 2, wherein the pumps are out of phase with each other bybetween 10 and 360 degrees.
 4. The system as described in claim 3,wherein the pumps are out of phase with each other by between 90 and 180degrees.
 5. A system for hemodynamic simulation, the system comprising:a vessel through which the fluid may be urged; a chamber in which thevessel is received, the chamber including a means for controllingpressure; a reservoir for retaining the fluid; a plurality of pumps influid communication with the fluid, one of the pumps urging the fluidthrough the vessel; and a means for controlling the pumps, wherein themeans for controlling the pumps is selected from the group consisting ofa cam mechanism; a multi-bar linkage mechanism; a solenoid; a steppermotor; an electric motor; a linear ball actuator; a belt-drivenactuator; and a chain-driven actuator.
 6. The system as described inclaim 1, further comprising a third pump, the third pump being connectedto the chamber, and wherein when the means for controlling pressure isapplied to the chamber, pressure is exerted on the vessel.
 7. The systemas described in claim 6, further comprising a means for adjusting thedownstream flow of the fluid between the vessel and the reservoir. 8.The system as described in claim 7, further comprising a steady flowpump, the steady flow pump being positioned between the reservoir andone of the pumps.
 9. The system as described in claim 8, furthercomprising a means for filtering noise, the means for filtering noisebeing positioned between the steady flow pump and the vessel.
 10. Thesystem as described in claim 5, wherein the means for controlling thepumps further comprises a computer system.
 11. The system as describedin claim 1, wherein the vessel is chosen from the group consisting ofmammalian blood vessels; models of mammalian blood vessels; endothelialcells; osteocytes; chondrocytes; and muscle cells.
 12. The system asdescribed in claim 1, wherein the plurality of pumps comprises: anupstream pump in fluid communication with the fluid, the upstream pumpurging the fluid through the vessel in a pushing manner; and adownstream pump in fluid communication with the fluid, the downstreampump being downstream of said upstream pump, the downstream pump urgingthe fluid through the vessel in a pulling manner.
 13. The system asdescribed in claim 1, wherein the plurality of pumps comprises: a pairof upstream pumps in fluid communication with the fluid.
 14. The systemas described in claim 1, wherein the plurality of pumps comprises: anupstream pump in fluid communication with the fluid, the upstream pumpurging the fluid through the vessel in a pushing manner; and an externalpump, the external pump being operatively connected to the chamber,wherein when the means for controlling pressure is applied to thechamber, pressure is exerted on the vessel.
 15. The system as describedin claim 1, wherein the plurality of pumps comprises: a downstream pumpin fluid communication with the fluid, the downstream pump urging thefluid through the vessel; and an external pump, the external pump beingoperatively connected to the chamber, wherein the means for controllingpressure is applied to the chamber, pressure is exerted on the vessel.16. A system for hemodynamic simulation, the system comprising: a fluid;a vessel through which the fluid may be urged; a chamber in which thevessel is received, the chamber including a means for controllingpressure; a reservoir for retaining the fluid; a plurality of pumps influid communication with the fluid, one of the pumps urging the fluidthrough the vessel; and a means for controlling the pumps comprising amotor, a cam, and a means for linking the pumps with each other, thepumps being operatively connected with the means for controlling thepumps, the means for linking the pumps being adjustable, the pumps beingout of phase with each other.
 17. The system as described in claim 16,wherein the plurality of pumps comprise: an upstream pump in fluidcommunication with the fluid, the upstream pump urging the fluid throughthe vessel in a pushing manner; and a downstream pump in fluidcommunication with the fluid, the downstream pump being downstream ofthe upstream pump, the downstream pump urging the fluid through thevessel in a pulling manner; a third pump operatively connected to themeans for controlling the pumps, the third pump being connected to thechamber, and wherein when the means for controlling pressure is appliedto the chamber, pressure is exerted on the vessel.
 18. The system asdescribed in claim 17, wherein the vessel is chosen from the groupconsisting of mammalian blood vessels; models of mammalian bloodvessels; endothelial cells; osteocytes; chondrocytes; and muscle cells.19. The system as described in claim 16, wherein the plurality of pumpscomprise: a pair of upstream pumps in fluid communication with thefluid.
 20. The system as described in claim 16, wherein the plurality ofpumps comprise: an upstream pump in fluid communication with the fluid,the upstream pump urging the fluid through the vessel in a pushingmanner; and an external pump, the external pump being operativelyconnected to the chamber, wherein when the means for controllingpressure is applied to the chamber, pressure is exerted on the vessel.21. The system as described in claim 16, wherein the plurality of pumpscomprise: a downstream pump in fluid communication with the fluid; thedownstream pump urging the fluid through the vessel in a pulling manner;an external pump, the external pump being operatively connected to thechamber, wherein when the means for controlling pressure is applied tothe chamber, pressure is exerted on the vessel.
 22. A method forsimulating biomechanical stimuli, the method comprising the steps of:providing a fluid; providing a vessel through which the fluid may beurged; providing a chamber for receiving the vessel therein, the chamberfurther including a means for controlling pressure, wherein said chamberis connected to a pump; providing an upstream pump in fluidcommunication with the fluid, the upstream pump urging the fluid throughthe vessel in a pushing manner; providing a downstream pump in fluidcommunication with the fluid, the downstream pump urging the fluidthrough the vessel in a pulling manner.
 23. The method as described inclaim 22, further comprising the step of applying the means forcontrolling pressure to the chamber, thereby exerting pressure on thevessel.
 24. The method as described in claim 22, wherein the vessel ischosen from the group consisting of mammalian blood vessels; models ofmammalian blood vessels; endothelial cells; osteocytes; chondrocytes;and muscle cells.
 25. The method as described in claim 23, furthercomprising the step of providing a means for controlling the pumps,wherein the upstream pump and the downstream pump are operativelyconnected with the means for controlling the pumps.
 26. The method asdescribed in claim 25, wherein the means for controlling the pumpscomprises a motor, a cam, and a means for linking the upstream pump withthe downstream pump.
 27. The method as described in claim 26, whereinthe means for linking the upstream pump and the downstream pump isadjustable, and wherein the upstream pump and the downstream pump areout of phase with each other.
 28. The method as described in claim 27,wherein the upstream pump and the downstream pump are out of phase witheach other by between 10 and 360 degrees.
 29. The method as described inclaim 28, wherein the upstream pump and the downstream pump are out ofphase with each other by between 90 and 180 degrees.
 30. The method asdescribed in claim 25, wherein the means for controlling the pumps isselected from the group consisting of a cam mechanism, a multi-barlinkage mechanism; a solenoid; a stepper motor; an electric motor; alinear ball actuator; a belt-driven actuator; and a chain drivenactuator.
 31. The method as described in claim 30, further comprisingthe step of providing a reservoir for retaining the fluid, the reservoirbeing in fluid communication with the vessel.
 32. The method asdescribed in claim 31, further comprising the step of providing a meansfor adjusting the downstream flow of the fluid between the vessel andthe reservoir.
 33. The method as described in claim 32, furthercomprising the step of providing a steady flow pump, the steady flowpump being positioned between the reservoir and the upstream pump. 34.The method as described in claim 33, further comprising the step ofproviding a means for filtering noise, the means for filtering noisebeing positioned between the steady flow pump and the vessel.
 35. Themethod as described in claim 34, wherein the means for controlling thepumps further comprises a computer system.
 36. The method as describedin claim 22, wherein the biomechanical stimuli are chosen from the groupconsisting of wall shear stress, circumferential strain, pulsatilepressure, transmural pressure, and biologically active agents.
 37. Amethod for hemodynamic simulation, the method comprising the steps of:providing a fluid; providing a vessel through which the fluid may beurged; providing a chamber in which the vessel is received, the chamberfurther including a means for controlling pressure; providing areservoir for retaining the fluid; providing a plurality of pumps influid communication with the fluid, wherein one of said pumps urges thefluid through the vessel; and providing a means for controlling thepumps, comprising a motor, a cam, and a means for linking the pumps witheach other, the pumps being operatively connected with the means forcontrolling the pumps, the means for linking the pumps being adjustable,the pumps being out of phase with each other.
 38. The system describedin claim 37, wherein the vessel is chosen from the group consisting ofmammalian blood vessels, models of mammalian blood vessels; endothelialcells; osteocytes; chondrocytes; and muscle cells.
 39. The method asdescribed in claim 37, further comprising: providing an upstream pump influid communication with the fluid; the upstream pump urging the fluidthrough the vessel in a pushing manner; and providing a downstream pumpin fluid communication with the fluid, the downstream pump beingdownstream of the upstream pump, the downstream pump urging the fluidthrough the vessel in a pulling manner; and providing a third pump, thethird pump operatively connected to the means for controlling the pumps,the third pump being connected to the chamber, and wherein when themeans for controlling the pressure is applied to the chamber, pressureis exerted on the vessel.
 40. The method as described in claim 37,further comprising: providing a pair of upstream pumps in fluidcommunication with the fluid.
 41. The method as described in claim 37,further comprising: providing an upstream pump in fluid communicationwith the fluid, the upstream pump urging the fluid through the vessel ina pushing manner; and providing an external pump the external pump beingoperatively connected to the chamber, wherein when the means forcontrolling pressure is applied to the chamber, pressure is exerted onthe vessel.
 42. The method as described in claim 37, further comprising:providing a downstream pump in fluid communication with the fluid, thedownstream pump urging the fluid through the vessel in a pulling manner;and providing an external pump, the external pump being operativelyconnected to the chamber, wherein when the means for controllingpressure is applied to the chamber, pressure is exerted on the vessel.43. The system of claim 1, wherein the fluid comprises tissue culturemedium, blood, physiological saline solution, or other bufferedsolution.
 44. The system of claim 16, wherein the fluid comprises tissueculture medium, blood, physiological saline solution, or other bufferedsolution.
 45. The method of claim 22, wherein the fluid comprises tissueculture medium, blood, physiological saline solution, or other bufferedsolution.
 46. A system for hemodynamic simulation, the systemcomprising: a vessel through which fluid may be urged; a chamber inwhich the vessel may be received, the chamber including a means forcontrolling pressure within the chamber and being connected to a pump; areservoir suitable for retaining a fluid; and a plurality of pumps, oneof said pumps being suitable for urging fluid through the vessel,wherein said pumps are operatively connected to the means forcontrolling the pumps, and wherein the means for controlling the pumpscomprises a motor, a cam, and a means for linking the pumps.